Project Summary X-linked transcription factor (TF) Foxp3 is a lineage specification factor for regulatory T (Treg) cells lineage, which is vital for limiting autoimmunity and inflammation. Foxp3 loss-of-function causes fatal human autoimmune IPEX syndrome and similarly deadly disease in mice. Increasing realization that Treg cells are playing an important role in diverse physiological and pathological processes, including autoimmunity, allergy, tissue repair, transplantation, metabolic inflammation, and degenerative disease, forms the basis for their potential use as cellular therapeutics. Foxp3 expression is essential for Treg cell differentiation and its continuous expression in differentiated Treg cells is required for their suppressor function. Thus, Foxp3-dependent transcriptional program lies at the core Treg cell biology. However, in-depth understanding of the Foxp3-mediated gene expression program and preceding events during Treg cell differentiation required for its execution is lacking. Our long-term goal in this application is to generate essential new knowledge of fundamental mechanistic underpinning of Treg biology ? its transcriptional control by Foxp3. Based on our recent studies, we will test a hypothesis that developmental history ?prepares? specific sets of chromatin sites in precursor cells for Foxp3 binding through activity of ?companion? TF preceding and subsequent to Foxp3 expression. This RO1 renewal application aims to identify key TFs guiding Treg differentiation and facilitating proper direct and indirect Foxp3-dependent control of gene expression defining Treg cell identity and function. In this proposal, we will leverage extensive natural genetic variation in laboratory C57Bl/6 (B6) and wild-derived CAST/EiJ (Cast) and SPRET/EiJ (Spret) mice, which represent different taxa separated over 1 million years ago, and take advantage of novel unique genetic tools recently generated in the lab to address major mechanistic questions in Treg cell biology. In Aim 1, we will investigate direct and indirect transcriptional regulation of gene expression by Foxp3 using natural genetic variation. In Aim 2, we will explore dynamics of regulation of gene expression by Foxp3 by studying temporal establishment of Foxp3-mediated transcriptional program. In Aim 3, we will identify stable and reversible components of Foxp3-dependent transcriptional program directly and indirectly controlled by Foxp3 and, thereby, reveal core Foxp3-dependent functional program in Treg cells. These studies will elucidate fundamental mechanisms of Foxp3-dependent regulation of gene expression in an unbiased and genetically controlled manner by leveraging power of genetics of evolutionary distant mice, high throughput genomics, and cutting- edge computational approaches combined with genome editing and biochemical and immunological analyses.